Gallium-68 DOTATATE Production with Automated PET Radiopharmaceutical Synthesis System: A Three Year Experience.

OBJECTIVES
Gallium-68 (Ga-68) is an ideal research and hospital-based PET radioisotope. Currently, the main form of Ga-68 radiopharmaceutical that is being synthesised in-house is Ga-68 conjugated with DOTA based derivatives. The development of automated synthesis systems has increased the reliability, reproducibility and safety of radiopharmaceutical productions. Here we report on our three year, 500 syntheses experience with an automated system for Ga-68 DOTATATE.


METHODS
The automated synthesis system we use is divided into three parts of a) servomotor modules, b) single use sterile synthesis cassettes and, c) a computerised system that runs the modules. An audit trail is produced by the system as a requirement for GMP production. The required reagents and chemicals are made in-. The Germanium breakthrough is determined on a weekly basis. Production yields for each synthesis are calculated to monitor the performance and efficiency of the synthesis. The quality of the final product is assessed after each synthesis by ITLC-SG and HPLC methods.


RESULTS
A total of 500 Ga-68 DOTATATE syntheses (>800 patient doses) were performed between March 2011 and February 2014. The average generator yield was 81.3±0.2% for 2011, 76.7±0.4% for 2012 and 75.0±0.3% for 2013. Ga-68 DOTATATE yields for 2011, 2012, and 2013 were 81.8±0.4%, 82.2±0.4% and 87.9±0.4%, respectively. These exceed the manufacturer's expected value of approximately 70%. Germanium breakthrough averaged 8.6×10(-6)% of total activity which is well below the recommended level of 0.001%. The average ITLC-measured radiochemical purity was above 98.5% and the average HPLC-measured radiochemical purity was above 99.5%. Although there were some system failures during synthesis, there were only eight occasions where the patient scans needed to be rescheduled.


CONCLUSION
In our experience the automated synthesis system performs reliably with a relatively low incident of failures. Our system had a consistent and reliable Ga-68 DOTATATE output with high labelling efficiency and purity. There is minimal operator intervention and radiation exposure. The system is GMP-compliant and has low maintenance and acceptable running costs. This system together with the recommended (68)Ge/(68)Ga generator is well suited for use in a hospital-based radiopharmacy.

deoxyglucose (FDG). F-18 is a cyclotronproduced radioisotope and, as a result, it requires an on-site cyclotron or access to a nearby cyclotron and subsequent delivery, costing both time and money. Generatorproduced radioisotopes, such as Ga-68, have a number of advantages that makes them attractive as hospital-based PET radioisotopes. Ga-68 has rapidly gained attention as one of the ideal research and hospital-based PET radioisotopes. It has the advantage of being produced on-site from a small wet-bed 68 Ge/ 68 Ga generator, so it can be made available within minutes as well as at much lower cost than other similar radioisotopes. It has a short half-life (67.7 minutes) making it suitable for human studies and low radiation dose to the patients. The trivalent nature of Gallium-68 also makes it well suited for radiolabelling the 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetra acetic acid (DOTA) compounds as well as labelling proteins and peptides (1).
Currently a general review of the reports published in the literature suggests that the main non-cyclotron produced PET radiopharmaceutical that is being synthesised in-house is Ga-68 conjugated with DOTA based derivatives, specifically for imaging over-expression of various subtypes of somatostatin receptors expressed by neuroendocrine tumours (NETs). The main radiopharmaceuticals being used for this application are DOTATATE, DOTATOC and DOTANOC, all of which are labelled via the DOTA chelator to Ga-68. Figure 1 shows examples of patient scans from our institution using both FDG and DOTATATE, demonstrating that these tumours can be either FDG or DOTATATE positive or negative in various combinations. Although the synthesis and purification of these radiopharmaceuticals can successfully be carried out manually in the laboratory, the use of automated synthesis systems (2), (3) is on the increase. These automated systems have clear advantages over the manual methods which have resulted in their increasing installation and use in hospitalbased radiopharmacies. Here we report on our three year experience of over 500 syntheses with an automated synthesis system for Ga-68 DOTATATE.

Methods
There are a number of reports in the literature using and detailing various automated synthesis systems. One of the automated systems that closely, but not exactly, resembles our system was reported by Decristoforo et al (4). Automated synthesis systems are generally set-up and adjusted to the specific needs of the institution. As a result over time, these setups evolve and are tweaked to improve productivity and become unique to the institution. The system used here was the Eckert & Ziegler Eurotope's Modular-Lab Pharm Tracer® automated synthesis system.
All materials and methods used were followed as instructed or later modified by Eckert & Ziegler Eurotope (Berlin, Germany) from whom the automated synthesis system and synthesis cassettes were obtained.

Reagents
As recommended by the manufacturer of the automated synthesis system, Eckert & Ziegler Eurotope (Berlin, Germany), all reagents were high purity pharmaceutical grade, unless stated otherwise. The exact grade as well as the source of the reagents used was their recommendation.
Water puriss p.a (FLUKA Trace SELECT® 95305) used in the preparation of the majority of reagents was obtained from Sigma-Aldrich, Australia. Hydrochloric acid (Ultrapure 30% HCl, 0. The DOTATATE, or DOTA-(Tyr 3 )-octreotate (where DOTA=1, 4, 7, 10 -tetraazacyclododecane -1, 4, 7, 10-tetraacetic acid) was obtained from Auspep, Victoria, Australia. The peptide is obtained as a 1 mg powder which is subsequently dissolved in 1 mL of water and used in 40 µl aliquots. Up till recently the DOTATATE was only available as a research grade peptide. However, since mid-2013 a Good Manufacturing Process (GMP) grade product has also been available which we now utilise.
The preparation of the required Ga-68 DOTATATE reagents and chemicals are shown in Table 1. This table is used as a quick instruction guide for the daily preparation of chemicals at our centre.

Synthesis Cassettes and Automation
The synthesis of DOTATATE is performed using the Eckert & Ziegler Eurotope's Modular-Lab Pharm Tracer® automated synthesis system designed specifically for the synthesis of radiopharmaceuticals. It can be separated into three parts. These are: a) the modules assembled according to the specific radiopharmaceutical being synthesised; b) the synthesis cassettes on which the appropriate chemicals, reagents, filters, columns, etc are attached; and c) the computerised system that runs the modules which in turn run the valves and syringes on the cassettes producing the required radiopharmaceutical. Since the system is computer-controlled, an audit trail is always maintained which is a requirement for GMP production runs.
There is a specific synthesis cassette for the production of Ga-68 DOTATATE as well as one for a simple elution of the Ga-68 generator. The system is also currently being used for the production of other radiopharmaceuticals, such as Lu-177 DOTATATE, using a specific synthesis cassette. The production cassettes are obtained from Eckert & Ziegler Eurotope. These cassettes are sterile and single-use.
As with the cassettes, there is a specific template computer program for each specific radiopharmaceutical synthesis process. These synthesis templates are also provided by Eckert & Ziegler Eurotope. These are subdivided into template programs for the cassette's pressure testing, terminal sterilisation filter testing, running the HPLC analysis and eluting the generator. The Modular-Lab software also allows programming and modifications to the  templates via a graphical user interface (GUI).

Generators
So far, on average one generator per year has been used during the three years of using the automated system. All three 68 Ge/ 68 Ga generators were obtained from Eckert & Ziegler Eurotope (Berlin, Germany). The first generator (IGG100-30M) used was a 1.11 GBq whereas the subsequent two (IGG100-50M) generators were 1.85 GBq. The latter two generators allowed for larger number of patient doses per synthesis and a longer "useful" life.
Due to the build-up of metal ions on the column, the generators are required to be eluted on a daily basis and within 24 hours prior to any DOTATATE synthesis. Therefore, the generator was eluted on the first day of the week and on non-synthesis days. The elution process involved using an elution cassette, 0.1 M HCl solution driven by the Modular-Lab system in approximately five minutes. The elution volume was 8 mL.

Automated Radiolabelling Process
The radiolabelling process is explained fully and in details in the instruction manual that is supplied with the Modular Lab Pharm Tracer® automated system. Briefly the process is summarised here.   The total volume of the final product is 8 mL.
The Modular Lab Pharm Tracer® software provided a graphical display of each step and progress of the synthesis (Figure 3). On completion, the synthesis process is saved on the computer for audit trail purposes ( Figure 4). The third phase of the synthesis involves testing of the integrity of the 0.22 µm filter. This is known as the Filter Pressure Test and is, again, driven by the Modular Lab Pharm Tracer® software. The only operator intervention required is the removal of the 0.22 µm filter and needle from the product vial and putting it into the waste bottle prior to the test. The test is performed by subjecting the filter to 200 kPa of pressure. If pressure is maintained above 100 kPa, the test is considered to have been successful and the product is sterile for patient use. However, if the pressure falls below 100 kPa, the test is considered to have been unsuccessful and there is a chance of the product being unsterile. To rectify this, the product needs to be drawn-up manually in a 10 mL syringe and passed through a new 0.22 µm filter into another sterile vial. To date, this has never occurred at our centre. On completion, the filter pressure test process log is saved on the computer for audit trail purposes. The trace produced by the software is identical to the ones produced for the Cassette Pressure Test ( Figure  2). After removing a 0.2 mL sample from the product for quality control purposes, the product is ready to be administered to the patient.

Analysis and Quality Control
The quality control of the resulting product is done by two methods of Instant Thin Layer Chromatography (ITLC) as well as High Pressure Liquid Chromatography (HPLC). These are performed using the 0.2 mL sample taken directly from the final Ga-68 DOTATATE product.

Instant Thin Layer Chromatography (ITLC)
The ITLC test is used to determine the percentage of Ga-68 DOTATATE, Ga-68 impurities, and Ga-68 colloid in the final product. The ITLC paper strips are counted in a laboratory gamma-counter (PerkinElmer Wizard 2 ® Automated Gamma Counter, PerkinElmer Downers Grove, IL USA) and

Ge-68 Breakthrough
The Ge-68 breakthrough is measured in an eluted sample, on a weekly basis, after a complete Ga-68 decay (>48 hours). The measurements are carried out in a laboratory gamma-counter (PerkinElmer Wizard 2 ® Automated Gamma Counter, PerkinElmer Downers Grove, IL USA) and the data entered into and calculations made using a Microsoft Excel version 2010 spread sheet.

Results
A total of approximately 500 Ga-68 DOTATATE syntheses were performed between March 2011 and April 2014 in our laboratory. All syntheses were carried out using the Eckert & Ziegler Eurotope's Modular Lab Pharm Tracer® automated synthesis system and software. All syntheses were performed in the Department of Nuclear Medicine, Royal North Shore Hospital in a purpose-built radiopharmaceutical laboratory.

Generators
Generator yields for the three years are shown in Figure 5. The percentage yield was calculated as the actual yield as a percentage of calculated as the actual yield as a percentage of the expected yield. The average generator yield for the first year was 81.3±0.2%. The yields were lower for second and third years at 76.7±0.4% and 75.0±0.3% corresponding to the larger generators. The expected yield, as indicated by the manufacturer, is approximately 70%.

Ga-68 DOTATATE Syntheses
Although the synthesis cassettes used for Ga-68 DOTATATE had the same overall design  Figure 6. Ga-68 DOTATATE synthesis yields for the automated synthesis system and specifications there were some minor modifications made by the manufacturer during the three years. These modifications were made as a part of ongoing improvements and quality control in response to user comments and feedback. Some of these modifications addressed the connections on the cassettes to increase reliability whereas some addressed the increase in product yield by, for example, increasing the volume of ethanol being used to elute the final product from the C18 cartridge. These resulted in the cassette failure rates dropping dramatically with time and are now very rare. In addition, these also resulted in the average synthesis yields increasing from 81.8 ± 0.4% in the first year to 82.2±0.4% (P=0.42) in the second year and 87.9±0.4% (P<0.001) in the third year ( Figure 5).
Due to the nature of the 68 Ge/ 68 Ga generator and the half-life of 271 days when new, enough activity can be obtained to synthesise sufficient Ga-68 DOTATATE for three patients per synthesis run (150 MBq to 200 MBq per dose). As the generator decays this is reduced to two patients after approximately one year and eventually to one patient. This reduces the costeffectiveness of the synthesis process requiring the purchase of a new generator. In order to predict how long the generator will be effective in producing enough Ga-68, a graph of final product activity against the age of generator ( Figure 6) was made and an equation of the line of best-fit was obtained. This took the average synthesis yields into account and produced a rough estimate as to when a new generator would be required to maintain the minimum required number of patients per synthesis run. months to provide enough product for more than one patient per run.

Germanium-68 Breakthrough
The germanium-68 breakthrough measured during the three years of use was constantly well below the recommended level of 0.001% of the total radioactivity. The actual average values were 8.6×10 -6 % of the total radioactivity.

Radiochemical Quality Control
The radiochemical purity of the final product was measured using ITLC as well as HPLC. The results demonstrated (Figure 7 These results were well above the minimum recommended value of 91% (5). The reason for the small decline in the ITLC-measured purity level during the three years has not been identified.  The actual Modular Lab System, with the exception of two occasions, performed flawlessly throughout the three years without any maintenance. However, the synthesis cassettes had relatively much higher failure rates. As a percentage of total number of syntheses carried out, these failure rates were very low (16.4%). From the total of 82 equipment failure events in the three year period, only eight (9.8%) led to a full synthesis failure and patients being deferred. This rate is also very low and all occurred in initial teething period. With more operator experience during the second and third years, some of the failures that could have led to full synthesis failures were successfully averted. In addition, feedback to the manufacturer led to a number of areas of concern being addressed -particularly those relating to the cassettes.

Radiochemical Purity By HPLC
The Ga-68 DOTATATE product radiochemical purity was measured using the ITLC as well as the HPLC techniques. Results showed that not only was the synthesised product above the minimum recommended level, but also was of high purity. There was, however, a marginal and gradual decline in ITLC values. We could not explain this decline. The very low variation in the product quality ( Figure  8, 9) demonstrated a consistent-quality production system that could reliably produce a very high quality product.

Conclusion
The automated synthesis system provides a reliable and straightforward approach to synthesising one particular short-lived PET radiopharmaceutical.
Eckert & Ziegler Eurotope's Modular-Lab Pharm Tracer® is an innovative automated synthesis system that performs reliably with a relatively low incident of failures. Our system had a consistent and reliable Ga-68 DOTATATE output with high purity exceeding the minimum recommended requirements. The system is GMP-compliant and has low maintenance and acceptable running costs.